Number, Proliferative Activity, and Expression of Thyroid Hormone Receptors in Dermal Fibroblasts in Mice with Changed Thyroid Status.

We studied the intensity of age-specific changes in the dermis (number and proliferative activity of fibroblasts) in mice with normal and experimentally changed level of thyroid hormones. Receptors of thyroid hormones, TR-α and TR-ß, in mouse dermal fibroblasts were identified by immunohistochemical methods. The relative expression of Thra, Thrb, and Dio2 genes was assessed by real-time PCR analysis. From the second to fifth month of life, the number of fibroblasts in the connective tissue layer of mouse skin decreased by 42.3%. The number of fibroblasts in the dermis of 5-month-old mice treated with Thyrozol significantly decreases by 25.9% (p<0.05), and vice versa, in mice receiving thyroxin this parameter increased by 4.7% in comparison with the control (p>0.05). TR-α and TR-ß were identified in dermal fibroblasts in all groups of mice. No differences in the content TR-α and Thra gene expression in 2- and 5-month-old mice of the control and experimental were revealed. TR-ß content in dermal fibroblasts of 2-month-old animals was maximum and exceeded this value in 5-month-old control mice by 25%. The number of these receptors decreased by 33.3% in mice treated with Thyrozol and increased by 25% in animals receiving thyroxin injection in comparison with the control. Relative expression of Thrb gene significantly increased only in mice treated with thyroxin. Comparative analysis of the relative expression of Dio2 gene revealed no differences between the experimental and control groups. Changes in the level of thyroid hormones, content of TR-ß, and relative Thrb gene expression contribute to agerelated shifts in the number and proliferative activity of mouse dermal fibroblasts.

[Thyroid hormone receptors in human skin during aging.]

This work was aimed to study levels of thyroid hormone receptors in human dermal fibroblasts from the development to deep aging. Skin specimens from human fetuses died antenatally from 20 to 40 weeks of pregnancy, humans died from different causes from birth to 85 years of life were used for the study. Total number of fibroblasts, percent of proliferating cells nuclear antigen (PCNA) positive dermal fibroblasts, expression of thyroid hormone receptors-α and -ß in dermal fibroblasts were examined. PCNA and thyroid hormone receptors were viewed immunohistochemically. A total number of fibroblasts in dermis were counting in sections stained with haematoxylin and eosin. Results showed that maximal levels of thyroid hormone receptors-α and -ß were observed from 20 to 40 weeks of pregnancy. The levels of thyroid hormone receptors-α and -ß were decreased from birth to 40 years of life. From 41 to 85 years, the levels of thyroid hormone receptors were approximately the same. A total number and percent of PCNA positive fibroblasts in dermis decreased with progression of age. Most sufficient age-dependent reduction in a total and PCNA positive number of dermal fibroblast was observed from antenatal until 40 years of life. Correlation analysis and one-way ANOVA showed that age-dependent decrease in the number of fibroblasts and retardation of their proliferation in human dermis is significantly associated with age-related decrease in the level of thyroid hormone receptors-α and -ß in dermal fibroblasts. Results allow to suggest that thyroid hormone receptors are involved in age-dependent decrease in the number and proliferation of fibroblasts in human dermis.

[The influence of metformin on age-related changes in the number and proliferation of dermal fibroblasts in mice.]

The goal of our work was to examine the effects of metformin on age-related changes in the number and proliferation of dermal fibroblasts in mice. The study of the dermis was carried out at five months' mice that received drinking water with metformin at concentrations 500 mg/l from two months (within 90 days). Five months' mice received drinking water without metformin and they were as a control. Material of two months' mice was also used in the work. We counted the total number of dermal fibroblasts and lobe of fibroblasts with positive coloration on Ki-67. The results showed that there has been a decrease in the total number of dermal fibroblasts by 42,3% from two months of mouse's life up to five months and lobe of fibroblasts with positive coloration on Ki-67 by 12%. The total number of dermal fibroblasts in five months' mice was lower for 13,4%by using metformin. The lobe of fibroblasts with positive coloration on Ki-67 was reduced by 27,3% in comparison with information of animals that have not received metformin. Thus, age-related reduction of dermal fibroblasts in mice is due to decline in their proliferative activity. Metformin has an inhibited impact on proliferation of dermal fibroblasts in mice.

[Age-related changes in the expression of lamin B receptors in fibroblasts of human dermis].

The aim of this work was to study lamin B receptors in human skin at different ages. Lamin B receptors, proliferating cells nuclear antigen (PCNA) were detected in sections of the skin by indirect immunohistochemistry. Our results showed that both portion of dermal fibroblasts with positive staining for lamin B receptors and intensity of staining of fibroblasts for lamin B receptors were maximal from birth to 20 years as compared to all other examined age-periods (from 20 weeks of pregnancy to 85 years old). General number of fibroblasts and number of fibroblasts with positive staining for PCNA in dermis were diminished with age. The most significant decrease in the number of fibroblasts and PCNA positive fibroblasts were observed from 20 years old. An increase in the level of lamin B receptors in dermal fibroblasts observed from birth to 20 years old may be regarded as one of start events leading to age-dependent decrease in the number of fibroblasts in human dermis.

[Age-related changes in the content of sirtuin 1 in fibroblasts of human dermis].

The goal of our work was to examine content of sirtuin 1 in human skin at different ages to uncover a role of sirtuin 1 in aging of human skin. Sirtuin 1, proliferating cells nuclear antigen (PCNA) were detected by indirect immunohistochemistry in sections of the skin of human fetuses died antenatally from 22 to 40 weeks of pregnancy and humans from birth to 85 years old, died from various causes. Our results showed that the level of sirtuin 1 in dermal fibroblasts was decreased from prenatal period to 85 years old. Both the number of fibroblasts and their proliferative activity were also decreased through life. Age-related decrease in sirtuin 1 content in dermal fibroblasts is statistically significant correlated with age-dependent decrease in proliferation. Therefore, lowering of sirtuin 1 content in dermal fibroblasts occurring with age can be regarded as a mechanism which leads to inhibition of proliferation of dermal fibroblasts.

[Age-related changes in the content of serine-arginine protein kinase 1 (SRPK1) in human dermis.]

The aim of our work was to examine content of serine-arginine protein kinase 1 (SRPK1) in human dermis at different ages (from 20 weeks of pregnancy to 85 years old). SRPK1, proliferating cells nuclear antigen (PCNA ), endothelial marker CD31 were detected in sections of the skin by indirect immunohistochemistry. Results showed, that content of SRPK1 in dermal fibroblasts was increased form antenatal period to 20 years of life followed by a decrease until 61-85 years period. SRPK1 content in dermal blood vessels is slowly gradually increased from antenatal period to 61-85 age interval. The number of fibroblasts and their proliferative activity, the number of CD31 positive blood vessels in dermis were decreased from antenatal period to 61-85 years period of life. Age-dependent decrease in SRPK1 in dermal fibroblasts from 20 years is associated with a reduction in the number and proliferative activity of fibroblasts. Age-related increase in SRPK1 content in dermal blood vessels is associated with a diminishing of the number of blood vessels. Hence, it can be supposed that SRPK1 has different actions on proliferation of differ components of dermis during aging.

Lamin A and lamin-associated polypeptide 2 (LAP-2) in human skin in the process of aging.

At present time, relationships between lamins and processes leading to aging are established. Mutations of genes of lamins lead to diseases, one of them is progeria. This disease is caused by violation of splaysing of lamin A gene and accumulation the farnezylated prelamin A (progerin) in the nucleus. LAP-2 is an important factor which regulates and stabilizes the lamin A. However, roles of lamin A and LAP-2 in behavior of population of dermal fibroblasts in relation to age were not examined. The aim of this research was to study A type lamin and LAP-2 in human skin at different ages. Lamin A and LAP-2 were detected in sections of the skin by indirect immunohistochemistry. The number of fibroblasts containing lamin A was gradually decreased from 90,4 to 76,9 % from 20 weeks of gestation to 85 years old. There were 32 % of dermal fibroblasts with positive staining for LAP-2 at the period from 20 weeks of gestation to 20 years old. From 21 to 40 years, 37,8 % of fibroblasts containing lamin A were found in the dermis. In age interval 41-85 years, 49-51 % of dermal fibroblasts had a positive staining for LAP-2. Content of lamin A in the nuclei of fibroblasts was almost constant from 20 weeks of gestation to 85 years old. Expression of LAP-2 in the nuclei of fibroblasts was reduced from birth to 20 years old but increased from 21 years old. Number of fibroblasts and PCNA+ fibroblasts in dermis was diminished with age. The most significant decrease in the number of fibroblasts was observed from 20 weeks of gestation to 20 years old. Results allow to assume the participation of lamin A and LAP-2 in triggering age-dependent decrease in the number of fibroblasts in the dermis in humans.

[Lamin B1 and lamin B2 in human skin in the process of aging].

The aim of this work was to study B type lamins in human skin at different ages. Lamins B1 and B2 were detected in sections of the skin by indirect immunohistochemistry. There were 62,3 % of dermal fibroblasts with positive staining for lamin B1 at the period from 20 to 40 weeks of gestation. From birth to 40 years, 41-42 % of fibroblasts containing lamin B1 were found in the dermis. In age interval from 41 to 85 years, 57-60 % of dermal fibroblasts had a positive staining for lamin B1. The number of fibroblasts containing lamin B2 was gradually decreased from 80,6 to 68,6 % from 20 weeks of gestation to 85 years old. Expression of lamin B1 in the nuclei of fibroblasts was reduced from birth to 40 years old. Content of lamin B2 in the nuclei of fibroblasts was almost constant from 20 weeks of gestation to 85 years old. Number of fibroblasts in dermis was diminished with age. The most significant decrease in the number of fibroblasts was observed from 20 weeks of gestation to 20 years old. Results allow to suggest the participation of lamin B1 in triggering age-dependent decrease in the number of fibroblasts in the dermis in humans.

[Age-related changes of the content of angiomatin and endostatin in human skin].

Human skin structures stained positively for angiomotin or endostatin were studied by indirect immunohistochemical method. Skin specimens from frontal surface of the lower part of the neck (from upper corner of standard autopsy skin incision) from human fetuses died antenatally from 20 to 40 weeks of pregnancy, humans who died from different causes from 1 day to 85 years of life were obtained at autopsy. Positive staining for angiomotin or endostatin in the skin was found in epidermal cells, fibroblasts, sweat and sebaceous glands, blood vessels of the dermis. Blood vessels stained positively for angiomotin were detected in skin samples in all ages. Age-dependent decrease in the content of angiomotin in blood vessels of the dermis was detected. Most prominent decrease in angiomotin content in dermal blood vessels was found in 61-85 years age-group. Endostatin positive blood vessels were also detected in skin samples of all ages. However, the intensity of staining for endostatin in dermal blood vessels was increased during aging. It can be proposed that changes in the content of angiomotin and endostatin yield a negative impact on angiogenesis in human skin during aging.

[Blood vessels in human dermis during aging].

A factor that potentially influences on skin aging is blood supply which determines global conditions for an organ or a tissue functioning, including skin. Scientific data on conditions of blood supply in the skin during aging are insufficient and contradictory. Therefore, this work was aimed to the study of age-related changes in the number of blood vessels in the human dermis. Blood vessels were visualized with immunohistochemical technique to two endothelial markers, as von Willebrand factor and antigen CD31. The results showed that von Willebrand factor and antigen CD31 are present in endothelial cells of blood vessels of dermis in all examined age periods, from 20 weeks of pregnancy to 85 yeas. Intensity of immunohistochemical staining to von Willebrand factor is enhanced during age. Intensity of staining to CD31 is not changed with age. The number of blood vessels positively stained either to von Willebrand factor or to CD31 in dermis was decreased gradually with age. A total number of fibroblasts in dermis decreased with age. The number of PCNA+ fibroblasts in dermis showing their proliferative activity was decreased with the progression of age. The decrease in the number of blood vessels is statistically associated with that in the general number of fibroblasts and proliferating fibroblasts. Hence, a factor that leads to aged decrease in the number of dermal fibroblasts is diminished blood supply, and actions targeted to enhancement of blood supply are to be in the basis of clinical approaches to prophylaxis and treatment aging changes of the skin.

[Biogenic amine level in the normal placental structures and in antiphospholipid syndrome].

Using the luminescent-histochemical methods, placenta was studied in 50 normal women with physiological pregnancy and in 35 patients in which the pregnancy progression was complicated by an antiphospholipid syndrome. The following bioamine-positive structures were identified in placenta: decidua, connective tissue and the blood vessels of chorionic plate, terminal villi containing capillaries, and syncytiotrophoblast. In antiphospholipid syndrome, the increase in histamine, catecholamine and serotonin luminescence intensity was detected in these structures. This increase was especially pronounced in histamine luminescence intensity.

Effect of toluene on bioamine-containing structures in the spleen.

The effect of toluene administered into the stomach on amino-containing structures in the spleen of random-bred albino mice was studied. It was shown by Falck-Hillarp method that 6 h after treatment the toxicant stimulated splenic mast cell population and inhibited other amino-containing structures. It is therefore suggested that in control mice the major role in bioamine metabolism in the spleen is played by granular fluorescent cells, while after poisoning, mast cells functioning as adapters acquire the primary role. The levels of catecholamines and serotonin in nervous and nonnervous structures peaked 1 week after poisoning and returned to normal after 4 weeks. Presumably, toluene suppresses the immunity starting from the second week after treatment.